The Study of anti-Malarials in Incomplete Lupus Erythematosus or SMILE is the first prevention study for systemic lupus erythematosus, randomizing 240 individuals with autoantibodies and at least one clinical feature of lupus to receive either hydroxychloroquine (HCQ) or placebo to assess the ability to delay or prevent the development of new clinical criteria or transition to classified SLE. Individuals are assessed quarterly for development of new serologic or clinical evidence of lupus, or other autoimmune rheumatic diseases, and standard samples are obtained. The ancillary studies proposed in this application will leverage the resources from this novel clinical trial with the addition of new critical samples required for analyses to test specific hypotheses of lupus pathogenesis and to identify potential mechanisms of how HCQ modifies the dysregulated autoimmune system in humans. The studies within this application focus on three critical hypotheses which have been established and confirmed in the field to be found in lupus after disease classification and will establish which of these pathways are critical to disease onset and pathogenesis. Autoantibodies are present in nearly all SLE patients and often occur years before the onset of clinical symptoms or disease classification. Using novel chip-based autoantigen and peptide arrays, experiments will assess the impact of HCQ on development of new autospecificities, and determine the degree and timing of diversification in response to accumulation of new clinical symptoms or SLE disease classification. Increased expression of interferon responsive genes is also common in SLE and associated with increased disease activity and autoantibody production. Results from retrospective analyses of serial samples from military and family collections of individuals who subsequently develop lupus support that interferon pathways are dysregulated before disease onset with greatest enhancement near classification. Interferon activity, whole blood gene signatures, soluble mediators and TLR/interferon stimulated responses will be assessed in the serial samples we will collect from this trial and analyzed in relation to the detailed protocol-driven clinical phenotyping. The influence of HCQ on these interferon responses, as well as the changes in these responses with the accumulation of additional lupus clinical features, will be assessed. Finally, autoantibody stimulated interferon production through netosis and the frequency and function of low density neutrophils will be tested on fresh serial samples to determine their temporal association with lupus symptom accrual. Finally, serologic sets of markers which have been identified to associate strongly with future onset of SLE in retrospective analyses will be tested with these newly available biomarker sets for the ability to predict accumulation of additional lupus criteria, as well as use the new data from this study to further refine the ability to identify individuals at the highest risk of SLE development for implementation of prevention strategies and/or further refined, biologically-relevant prevention trials.